TWI456064B - Fermentative production of nonvolatile microbial metabolites in solid form - Google Patents

Fermentative production of nonvolatile microbial metabolites in solid form Download PDF

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TWI456064B
TWI456064B TW095132992A TW95132992A TWI456064B TW I456064 B TWI456064 B TW I456064B TW 095132992 A TW095132992 A TW 095132992A TW 95132992 A TW95132992 A TW 95132992A TW I456064 B TWI456064 B TW I456064B
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fermentation broth
carbon atoms
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Markus Pompejus
Stephan Freyer
Markus Lohscheidt
Oskar Zelder
Matthias Boy
Edzard Scholten
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Basf Ag
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Claims (24)

一種藉由以糖為基礎之微生物發酵作用生產至少一種非揮發性固態微生物代謝物之方法，其中該方法中生產所需代謝物之微生物菌株係使用具有以液體培養基總重量計之20%重量比以上單醣含量之含糖液體培養基培育，藉此獲得一發酵液，該發酵液含有所欲代謝物及用於生產含糖液體培養基之澱粉原料中之非澱粉成分，且隨後該含有該代謝物之發酵液之揮發性成分係自該發酵液移除，直至殘餘水分含量為以該等發酵液之固體成分總乾重計之0.2至20%重量比，而未預先分離掉該發酵液之不溶性成分，其中該含糖液體培養基係以包含以下之步驟製備：a1)製備研磨基料，其係藉由研磨選自穀粒之澱粉原料；及a2)在至少一種澱粉液化酶存在下，在水性液體中液化該研磨基料，繼之使用至少一種糖化酶進行糖化作用，藉以獲得具有20%重量比以上之單醣含量之含糖液體培養基，其中該含糖液體培養基亦包含至少50%重量比之該澱粉原料之非澱粉固體成分；其中，對於該液化目的而言，以所使用研磨基料之總重量計，至少40%重量比之研磨基料在該液化期間係連續性地或批次性地添加至該水性液體中。 A method for producing at least one non-volatile solid microbial metabolite by sugar-based microbial fermentation, wherein the microbial strain producing the desired metabolite in the method uses 20% by weight based on the total weight of the liquid medium The above monosaccharide-containing sugar-containing liquid medium is cultured, thereby obtaining a fermentation liquid containing the desired metabolite and the non-starch component in the starch raw material for producing the sugar-containing liquid medium, and then the metabolite is contained The volatile component of the fermentation broth is removed from the fermentation broth until the residual moisture content is 0.2 to 20% by weight based on the total dry weight of the solid content of the fermentation broth without pre-separating the insolubleness of the fermentation broth a composition, wherein the sugar-containing liquid medium is prepared by the steps of: a1) preparing a mill base by grinding a starch material selected from the group consisting of: and a2) in the presence of at least one starch liquefaction enzyme, at a water Liquefying the ground base in a liquid, followed by saccharification using at least one saccharifying enzyme to obtain a sugar content having a monosaccharide content of 20% by weight or more a body medium, wherein the sugar-containing liquid medium further comprises at least 50% by weight of the non-starch solid component of the starch material; wherein, for the purpose of liquefaction, at least 40% by weight based on the total weight of the mill base used The ground base is continuously or batchwise added to the aqueous liquid during the liquefaction. 如請求項1之方法，其包含a)如步驟a1)及a2)中所述，製備具有20%重量比以上之 單醣含量之含糖液體培養基，其中該含糖液體培養基亦包含該澱粉原料之非澱粉固體成分；b)在發酵中使用該含糖液體培養基，以便生產該(等)非揮發性代謝物；及c)藉由移除該發酵液之至少某些揮發性成分，從該發酵液中獲得該(等)非揮發性固態代謝物及該澱粉原料之至少部分的非澱粉固體成分。 The method of claim 1, which comprises a) preparing 20% by weight or more as described in steps a1) and a2) a monosaccharide-containing sugar-containing liquid medium, wherein the sugar-containing liquid medium further comprises a non-starch solid component of the starch material; b) using the sugar-containing liquid medium in fermentation to produce the (etc.) non-volatile metabolite; And c) obtaining the (etc.) non-volatile solid metabolite and at least a portion of the non-starch solid component of the starch material from the fermentation broth by removing at least some of the volatile components of the fermentation broth. 如請求項1或2之方法，其中該研磨基料係在至少一種α-澱粉酶存在下在水性液體中液化，並隨後使用至少一種葡糖澱粉酶加以糖化。 The method of claim 1 or 2, wherein the mill base is liquefied in an aqueous liquid in the presence of at least one alpha-amylase and subsequently saccharified using at least one glucoamylase. 如請求項3之方法，其中該至少一種α-澱粉酶之部分係在步驟a2)之液化期間添加至該水性液體中。 The method of claim 3, wherein the portion of the at least one alpha-amylase is added to the aqueous liquid during liquefaction of step a2). 如請求項1或2之方法，其中該穀物係選自玉米、黑麥、黑小麥及小麥穀粒。 The method of claim 1 or 2, wherein the cereal is selected from the group consisting of corn, rye, triticale, and wheat grain. 如請求項1或2之方法，其中步驟a1)之研磨期間所獲得之該研磨基料包含至少50%重量比之粗粉顆粒，該等粗粉顆粒具有100微米以上之粒度。 The method of claim 1 or 2, wherein the mill base obtained during the grinding of step a1) comprises at least 50% by weight of coarse powder particles having a particle size of 100 microns or more. 如請求項1或2之方法，其中該研磨基料在步驟a2)中之液化及糖化作用係在使該液體培養基之黏度不超過20Pas之方式進行。 The method of claim 1 or 2, wherein the liquefaction and saccharification of the mill base in step a2) is carried out in such a manner that the viscosity of the liquid medium does not exceed 20 Pas. 如請求項1或2之方法，其中在該液化期間所添加之該研磨基料總量之至少25%重量比係在高於該研磨基料中所存在之澱粉之膠凝溫度的溫度下添加。 The method of claim 1 or 2, wherein at least 25% by weight of the total amount of the mill base added during the liquefaction is added at a temperature above the gelation temperature of the starch present in the mill base. . 如請求項1或2之方法，其中具有30%重量比以上單醣含 量之含糖液體培養基係經製備。 The method of claim 1 or 2, wherein the method comprises a monosaccharide content of 30% by weight or more A quantity of the sugar-containing liquid medium is prepared. 如請求項1或2之方法，其中至少一種植酸酶係在該發酵步驟之前添加至該含糖液體培養基中。 The method of claim 1 or 2, wherein at least one phytase is added to the sugar-containing liquid medium prior to the fermentation step. 如請求項1或2之方法，其中該(等)所生產之非揮發性代謝物係選自：有機單羧酸、二羧酸及三羧酸，其視情況具有附著於其之羥基且具有3至10個碳原子；蛋白型及非蛋白型胺基酸、嘌呤鹼基、嘧啶鹼基；核苷、核苷酸、脂質；飽和及不飽和脂肪酸；具有4至10個碳原子之二醇、具有3或3個以上羥基之更高官能度醇、具有至少4個碳原子之更長鏈醇、碳水化合物、芳族化合物、維生素、原維生素、輔因子、營養品、蛋白質、類胡蘿蔔素、具有3至10個碳原子之酮、內酯、生物聚合物及環糊精。 The method of claim 1 or 2, wherein the non-volatile metabolite produced by the (etc.) is selected from the group consisting of: an organic monocarboxylic acid, a dicarboxylic acid, and a tricarboxylic acid, optionally having a hydroxyl group attached thereto and having 3 to 10 carbon atoms; proteinaceous and non-protein amino acids, purine bases, pyrimidine bases; nucleosides, nucleotides, lipids; saturated and unsaturated fatty acids; diols having 4 to 10 carbon atoms a higher functionality alcohol having 3 or more hydroxyl groups, a longer chain alcohol having at least 4 carbon atoms, a carbohydrate, an aromatic compound, a vitamin, a provitamin, a cofactor, a nutrient, a protein, a carotenoid Ketones, lactones, biopolymers and cyclodextrins having 3 to 10 carbon atoms. 如請求項1或2之方法，其中該等所製備之非揮發性代謝物係選自酶、胺基酸、維生素、雙醣、具有3至10個碳原子之脂族單羧酸及二羧酸、具有3至10個碳原子之脂族羥基羧酸、具有3至10個碳原子之酮、具有4至10個碳原子之烷醇及具有3至10個碳原子之烷二醇。 The method of claim 1 or 2, wherein the non-volatile metabolites prepared are selected from the group consisting of an enzyme, an amino acid, a vitamin, a disaccharide, an aliphatic monocarboxylic acid having 3 to 10 carbon atoms, and a dicarboxylic acid. An acid, an aliphatic hydroxycarboxylic acid having 3 to 10 carbon atoms, a ketone having 3 to 10 carbon atoms, an alkanol having 4 to 10 carbon atoms, and an alkanediol having 3 to 10 carbon atoms. 如請求項1或2之方法，其中該等微生物係選自可生產至少一種以下代謝物之天然或重組微生物：酶、胺基酸、維生素、雙醣、具有3至10個碳原子之脂族單羧酸及二羧酸、具有3至10個碳原子之脂族羥基羧酸、具有3至10個碳原子之酮、具有4至10個碳原子之烷醇及具有3至10個碳原子之烷二醇。 The method of claim 1 or 2, wherein the microorganisms are selected from natural or recombinant microorganisms capable of producing at least one of the following metabolites: an enzyme, an amino acid, a vitamin, a disaccharide, an aliphatic having 3 to 10 carbon atoms a monocarboxylic acid and a dicarboxylic acid, an aliphatic hydroxycarboxylic acid having 3 to 10 carbon atoms, a ketone having 3 to 10 carbon atoms, an alkanol having 4 to 10 carbon atoms, and having 3 to 10 carbon atoms Alkanediol. 如請求項13之方法，其中該等微生物係選自：棒狀桿菌屬(Corynebacterium)、芽胞桿菌屬(Bacillus)、棉阿舒囊黴屬(Ashbya)、埃希氏菌屬(Escherichia)、麯黴屬(Aspergillus)、產鹼桿菌屬(Alcaligenes)、放線桿菌屬(Actinobacillus)、厭氧螺菌屬(Anaerobiospirillum)、乳酸桿菌屬(Lactobacillus)、丙酸桿菌屬(Propionibacterium)、梭菌屬(Clostridium)及根黴屬(Rhizopus)，其中尤其為以下菌株：麩胺酸棒桿菌(Corynebacterium glutamicum)、枯草芽孢桿菌(Bacillus subtilis)、棉阿舒囊黴(Ashbya gossypii)、大腸桿菌(Escherichia coli)、黑麯黴(Aspergillus niger)、協腹產鹼桿菌(Alcaligenes latus)、產琥珀酸厭氧螺菌(Anaerobiospirillum succiniproducens)、琥珀酸放線桿菌(Actinobacillus succinogenes)、德氏乳酸桿菌(Lactobacillus delbrückii)、萊希曼氏乳桿菌(Lactobacillus leichmannii)、阿拉伯糖丙酸桿菌(Propionibacterium arabinosum)、Propionibacterium schermanii、費氏丙酸桿菌(Propionibacterium freudenreichii)、丙酸梭狀芽孢桿菌(Clostridium propionicum)、丙酮丁醇梭桿菌(Clostridium acetobutlicum)、Clostridium formicoaceticum、米根黴(Rhizopus oryzae)及少根根黴(Rhizopus arrhizus)。 The method of claim 13, wherein the microorganisms are selected from the group consisting of: Corynebacterium, Bacillus, Ashbya, Escherichia, Aspergillus Aspergillus, Alcaligenes, Actinobacillus, Anaerobiospirillum, Lactobacillus, Propionibacterium, Clostridium And Rhizopus, especially the following strains: Corynebacterium glutamicum, Bacillus subtilis, Ashbya gossypii, Escherichia coli, black Aspergillus niger, Alcaligenes latus, Anaerobiospirillum succiniproducens, Actinobacillus succinogenes, Lactobacillus delbrückii, Lehman Lactobacillus leichmannii, Propionibacterium arabinosum, Propionibacterium schermanii, Fischerella Coli (Propionibacterium freudenreichii), propionic acid Clostridium (Clostridium propionicum), Clostridium acetobutylicum (Clostridium acetobutlicum), Clostridium formicoaceticum, Rhizopus oryzae (Rhizopus oryzae), and Rhizopus arrhizus (Rhizopus arrhizus). 如請求項1或2之方法，其中該發酵液中所存在之不超過30%重量比之固體係在移除該發酵液之揮發性成分之前 移除。 The method of claim 1 or 2, wherein no more than 30% by weight of the solids present in the fermentation broth is prior to removing volatile components of the fermentation broth Remove. 如請求項1或2之方法，其中該發酵液之液相係經移除而未預先分離掉該發酵液之不溶性成分，且該代謝物與該發酵液之所有不溶性成分的全部一起獲得。 The method of claim 1 or 2, wherein the liquid phase of the fermentation broth is removed without previously separating the insoluble component of the fermentation broth, and the metabolite is obtained together with all of the insoluble components of the fermentation broth. 如請求項1或2之方法，其中該至少一種非揮發性代謝物係以固態型式與所有不溶性成分的全部一起從該發酵液中獲得，而未預先移除該發酵液之不溶性成分。 The method of claim 1 or 2, wherein the at least one non-volatile metabolite is obtained from the fermentation broth together with all of the insoluble components in a solid form without removing the insoluble component of the fermentation broth in advance. 如請求項1或2之方法，其中該發酵液之揮發性成分自該發酵液移除，直至殘餘水分含量為以該等固體成分總乾重計之0.2至20%重量比，較佳地為1至15%重量比，及特佳地為5至10%重量比。 The method of claim 1 or 2, wherein the volatile component of the fermentation broth is removed from the fermentation broth until the residual moisture content is from 0.2 to 20% by weight, based on the total dry weight of the solid components, preferably 1 to 15% by weight, and particularly preferably 5 to 10% by weight. 如請求項1或2之方法，其中該發酵液係經噴霧乾燥、流化床乾燥或冷凍乾燥，以移除該等揮發性成分。 The method of claim 1 or 2, wherein the fermentation broth is spray dried, fluidized bed dried or freeze dried to remove the volatile components. 如請求項19之方法，其中一或多種乾燥佐劑係經使用。 The method of claim 19, wherein one or more of the drying adjuvants are used. 一種代謝物之固體調配物，其可藉由如請求項1至20中任一項之方法獲得，其包含：A)>10至80%重量比之至少一種非揮發性代謝物；B)1至50%重量比之生物質量，其係來自生產該非揮發性代謝物之發酵作用；C)1至50%重量比之來自該發酵液之該澱粉原料之非澱粉固體成分；及D)以組份A、B及C之總重量計之0至400%重量比之習知調配佐劑；A、B與C重量份總和為100%重量比。 A solid formulation of a metabolite obtainable by the method of any one of claims 1 to 20, comprising: A) > 10 to 80% by weight of at least one non-volatile metabolite; B) 1 To a 50% by weight biomass derived from the fermentation of the non-volatile metabolite; C) from 1 to 50% by weight of the non-starch solids of the starch material from the fermentation broth; and D) A conventional blending adjuvant of 0 to 400% by weight based on the total weight of parts A, B and C; the sum of parts by weight of A, B and C is 100% by weight. 如請求項21之調配物，其包含以該調配物總重量計至少5%重量比之膳食纖維。 The formulation of claim 21, which comprises at least 5% by weight of dietary fiber based on the total weight of the formulation. 一種如請求項21或22之調配物之用途，其係用於人類或動物營養。 Use of a formulation as claimed in claim 21 or 22 for human or animal nutrition. 一種如請求項21或22之調配物之用途，其係用於處理紡織品、皮革、纖維素、紙或表面。 Use of a formulation as claimed in claim 21 or 22 for the treatment of textiles, leather, cellulose, paper or surfaces.
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